Glycogen debranching enzyme (GDE) is a multifunctional enzyme acting as 1,4-α-D-glucan: 1,4-α-D-glucan 4-α-D-glycosyltransferase (E.C 2.4.1.25) and amylo-1,6-glucosidase (E.C 3.2.1.33) in glycogen degradation. The two activities of the debranching enzyme are believed to reside at separate sites on a single polypeptide chain with a molecular mass of 174 kDa. The structure-function domain has not been studied in detail (Chen and Burchell, Glycogen storage disease. In: The Metabolic and Molecular Bases of Inherited Disease, C. R. Scriver et al, eds., 7th Edition McGraw-Hill/New York, pp. 935-965 (1995); Bates et al, FEBS Lett. 58:181-185 (1975); Gillard et al, Biochemistry 16:3978-3987 (1977); Chapter 71 Kishnani P S, Koeberl D, Chen Y T. Glycogen Storage Diseases, in The Online Metabolic & Molecular Bases of Inherited Disease, Valle D, Beaudet A L, Vogelstein B, Kinzler K W, Antonarakis S E, Ballabio A, Scriver C R, Sly W S, Childs B, Editors (2008)). The predominant form of cDNA that encodes human debrancher has a 4596 bp coding region and a 2371 bp 3′ nontranslated region (Yang et al, J. Biol. Chem. 267:9294-9299 (1992)). Tissue specific debrancher mRNAs exist. These isoforms differ at the 5′ nontranslated region and are believed to be generated by differential RNA transcription and splicing from a single debrancher gene (Bao et al, Gene 197:389-398 (1997)). The human gene is localized to chromosome 1p21 (Yang-Feng et al, Genomics 13:931-934 (1992)). The genomic structure of the human GDE gene has been determined and consists of 35 exons spanning ˜85 kb of DNA.
Debranching enzyme, together with phosphorylase, is responsible for complete degradation of glycogen. Liver and muscle are the two major organs most active in glycogen metabolism. The primary function of glycogen in these organs is different. In muscle, glycogen provides a local fuel store for short-term energy consumption. In liver, it maintains glucose homeostasis.
Genetic deficiency of glycogen debranching enzyme (Glycogen Storage Disease-type III, GSD-III) causes an incomplete glycogenolysis resulting in accumulation of glycogen with abnormally short outer chain in various organs. The commonly affected organs in GSD-III are liver, skeletal muscle and heart. The disease is characterized by hepatomegaly, hypoglycemia, short stature, variable myopathy and cardiomyopathy. Patients with this disease vary remarkably, both clinically and enzymatically (Markowitz et al, Gastroenterology 105:1882-1885 (1993); Shen et al, J. Clin. Invest. 98:352-357 (1996); Telente et al, Annals. Intern. Med. 120:218-226 (1994)). Most patients have disease involving both liver and muscle (type IIIa), some patients (˜15% of all GSD-III patients) have only liver involvement (type IIIb), and, in rare cases, there is a selective loss of only one of the two GDE activities (glucosidase, (type IIIc) or transferase (type IIId)). Liver symptoms in GSD-III can improve with age and may disappear after puberty. Overt liver cirrhosis has been seen in some patients, some have developed hepatocellular carcinoma. Muscle weakness, though minimal during childhood, may become predominant in adults with onset in the third or fourth decade. These patients have slowly progressive proximal weakness and distal muscle wasting and some patients become wheelchair bound. Even within the subgroup of patients who develop myopathy/cardiomyopathy there is clinical variability. Some patients have asymptomatic cardiomyopathy, some have early symptomatic cardiomyopathy leading to death, and some have only muscle and no apparent heart involvement. An abnormal electrocardiogram (ECG) with ventricular hypertrophy is a frequent finding and does not correlate with clinical severity. Normal serum creatine kinase levels do not rule out muscle enzyme deficiency. The biochemical subtypes do not predict clinical severity.
There appears to be no correlation between the amount of debrancher protein and clinical severity (Yang et al, Am. J. Hum. Genet. 41:A28 (1992)). To predict accurately at initial diagnosis whether myopathy or cardiomyopathy may occur, one must determine whether debranching enzyme activity is deficient in muscle (Chen and Burchell, Glycogen storage disease. In: The Metabolic and Molecular Bases of Inherited Disease, C. R. Scriver et al, eds., 7th Edition McGraw-Hill/New York, pp. 935-965 (1995)). It appears that muscle disease will not develop in patients with GDE activity retained in muscle (Coleman et al, Annals of Internal Medicine 116:896-900 (1992)).
The variable phenotype is, in part, explained by differences in tissue-specific expression of the defective enzyme. As pointed out above, in type IIIa, enzyme is deficient in both liver and muscle, in IIIb there is enzyme deficiency only in liver. Unlike phosphorylase, which has tissue-specific isoenzymes encoded by different genes, at the protein level and at the molecular level it appears that there are no tissue-specific GDE isoenzymes in different tissues. Until now, it has not been understood in GSD-III how a single GDE gene, normally expressed in all tissues, can change expression in different tissues (Chen and Burchell, Glycogen storage disease. In: The Metabolic and Molecular Bases of Inherited Disease, C. R. Scriver et al, eds., 7th Edition McGraw-Hill/New York, pp. 935-965 (1995)). Two mutations (17delAG and G6X), both located in exon 3 at amino acid codon 6, are exclusively found in the GSD-IIIb (Shen et al, J. Clin. Invest. 98:352-357 (1996)) suggesting that exon 3 is important in controlling tissue-specific expression of the GDE gene.
Histology of the liver in these patients is characterized by a universal distension of hepatocytes by glycogen and the presence of fibrous septa. Electron microscopy studies on muscle specimens have shown presence of accumulated glycogen beneath the sarcolemma and between myofibrils; the excess glycogen not only disperses in the cytoplasm, but is also seen in the lysosomes (Cornelio et al, Arch. Neurol. 41:1027-1032 (1984), Miranda et al, Ann. Neurol. 9:283-288 (1981)).
The detailed structural biology of GDE is not known, although several functional domains of glycogen debranching enzyme have been proposed from enzymological studies and sequence comparison to other enzymes with similar catalytic function (Yang et al, J. Biol. Chem. 267:929409299 (1992), Liu et al, Archives of Biochemistry and Biophysics 306:232-239 (1993), Liu et al, Biochemistry 34:7056-7061 (1995), Jespersen et al, Journal of Protein Chemistry 12(6):791-805 (1993)). A region at the COOH-terminal of the debranching enzyme could be a candidate for glycogen binding site (Yang et al, J. Biol. Chem. 267:9294-9299 (1992)), and 4 regions at N-terminal half of the enzyme bear sequence homology to the catalytic sites identified or proposed in other amylolytic enzymes (Liu et al, Archives of Biochemistry and Biophysics 306:232-239 (1993), Jespersen et al, Journal of Protein Chemistry 12(6):791-805 (1993)). Aspartate at position 549 has been identified as the catalytic nucleophile in the transferase site of rabbit muscle glycogen debranching enzyme (Braun et al, Biochemistry 35:5458-5463 (1996)).
Currently there is no effective treatment for the disease. Hypoglycemia can be controlled by frequent meals high in carbohydrates with cornstarch supplements or nocturnal gastric drip feedings. Patients with myopathy have been given diets high in protein during the daytime plus overnight enteral infusion. In some patients, transient improvement in symptoms has been documented but there are no long-term data demonstrating that the high protein diet prevents or treats the progressive myopathy (Chen and Burchell, Glycogen storage disease. In: The Metabolic and Molecular Bases of Inherited Disease, C. R. Scriver et al, eds., 7th Edition McGraw-Hill/New York, pp. 935-965 (1995)). The progressive myopathy and/or cardiomyopathy is a major cause of morbidity in adults and patients with progressive liver cirrhosis and hepatic carcinoma have been reported. While gene therapy delivery of a normal, functional gene into the diseased organ could ultimately cure the disease, an ideal gene delivery vehicle that is reliable is currently not available. There is no living animal model for this disease. Dogs affected with GSD-III have been reported (Gregory et al, J. Vet. Intern. Med. 21(1):40-46(2007)), and a breeding colony is currently being established.
Enzyme replacement therapy has been effective in diseases in which the responsible enzymes/proteins exert their functions in extracellular fluids, such as adenosine deaminase deficiency, hemophilia, and α1-antitrypsin deficiency, or in a lysosomal location such as a lysosomal storage disease. Enzyme replacement has not been explored in diseases in which the defective enzyme is present in cytosol (such as the debranching enzyme in GSD-III), presumably due to the lack of an efficient and specific cellular uptake mechanism that delivers exogenous enzyme across the plasma membrane into the cytoplasm. Liposomes can fuse with plasma membrane and deliver their content, however, the use of liposomes is compromised by lack of organ-specific tropism and clearance by the reticulo-endothelial system (Mumtaz et al, Glycobiology 1(5):505-510 (1991)). For an effective treatment for GSD-III, the enzyme should be able to target muscle and heart as well as liver.
Cytoplasmic glycogen is normally digested by phosphorylase and debranching enzyme; excess glycogen taken up by lysosomes through autophagy can be digested by lysosomal acid α-glucosidase (GAA). Deficiency of debranching enzyme activity results in massive accumulation of glycogen having abnormally short outer branches. The excess glycogen in GSD-III resides not only in the cytoplasm but also in the lysosomes (cytoplasm>lysosome) (Cornelio et al, Arch. Neurol. 41:1027-1032 (1984), Miranda et al, Ann. Neurol. 9:283-288 (1981)). This suggests that the “normal” GAA activity in GSD-III may not be sufficient to digest all the excess glycogen. GAA is a lysosomal exo 1,4-α-D-glucosidase that hydrolyzes both α-1,4 and α-1,6 linkages of glycogen and can completely digest glycogen with and without abnormally short outer branches (Onodera et al, J. Biochem. 116:7-11 (1994)). GAA thus acts on glycogen with abnormally short outer branches such as accumulates in GSD-III. As this glycogen in GSD-III accumulates both in cytoplasm and lysosomes, providing more GAA may help to digest lysosomal glycogen and hence also cytoplasmic glycogen. It is postulated that, as the lysosomes are cleared of glycogen, glycogen from the cytoplasm shuffles into them thus decreasing the total amount of accumulated glycogen in GSD-III patients.
Deficiency of GAA causes Pompe disease (type II glycogen storage disease), a fatal metabolic myopathy with accumulation of glycogen in lysosome and cytoplasm (lysosome>cytoplasm) (Hirschhorn, Glycogen Storage Disease Type II: Acid α-glucosidase (Acid Maltase) Deficiency. In: The Metabolic and Molecular Bases of Inherited Disease, C. R, Scriver et al, eds., 7th Edition McGraw-Hill/New York, pp. 2443-2464 (1995)). Enzyme replacement therapy with mannose-6-Phosphate (man-6-P)-rich precursor recombinant human GAA results in efficient man-6-P receptor mediated endocytosis of the enzyme followed by reduction of both lysosomal and cytoplasmic glycogen in fibroblasts (Van Hove et al, Proc. Natl. Acad. Sci. 93:65-70 (1996)). In vivo, this enzyme targets heart and muscle as well as liver and spleen following intravenous injection in animals and in human Pompe patients. The rapid clearance of glycogen in Pompe fibroblasts when cultured in glucose-free medium suggests a ready mobilization of glycogen from both lysosomal and cytoplasmic compartments (Van Hove et al, Proc. Natl. Acad. Sci. 93:65-70 (1996), DiMauro et al, Pedatr. Res. 7:739-744 (1973)). It is contemplated that cytoplasmic glycogen continuously shuffles through lysosomes by autophagy for degradation.
The present invention results, at least in part, from the realization that administered GAA can reduce lysosomal glycogen in GSD-III patients and ultimately also reduce cytoplasmic glycogen. Some of the administered GAA may also go directly into the cytosol and reduce the glycogen. The invention provides a method of treating GSD-III (as well as GSD-IV, -VI, -IX, XI and cardiac glycogenosis due to AMP-activated protein kinase gamma subunit 2 deficiency) based on the use of GAA.